The invention relates generally to a method and apparatus for measuring thickness of transparent films and is used for measuring thickness of transparent, partially reflective films. For example, the invention can be used in blood analysis based, inter alia, on the quantitative evaluation of the intensities of a fluorescence radiation of a blood film. To perform the quantitative evaluation accurately, it is important to know the thickness of the blood film.
For example, in blood analysis, the blood is present in a special single-use microcuvette made of glass or plastic or a glass/plastic combination. Production-related tolerances in the dimensions of a single-use microcuvette make it necessary to measure the thickness of the blood film at different points of the already filled single-use microcuvette.
It would be possible to monitor the thickness tolerances of a single-use microcuvette, for example, with a mechanical feeler. The thickness of the blood film is determined, however, by the internal dimensions of a single-use microcuvette, which may have tolerance deviations different from those of the outside dimensions. But because the feeler can sense only the outside dimensions, it is unsuitable for determining the thickness of the blood film.
Another method for monitoring thickness tolerances of a single-use microcuvette involves interferometric measurements of the blood film. This is done, for example, by using an interferometer arrangement in a reflected-light microscope where the illumination light is propagating out of a spectrometer.
A filled single-use microcuvette is placed on the object stage of the microscope to be illuminated by the interferometer beam. The microscope illumination beam is reflected and interfered with all the interfaces of the single-use microcuvette. The measurement is performed for various wavelengths of the illumination light provided by the spectrometer. If the refractive index is known, the thickness of the individual films can be determined from the interference values as a function of the wavelength. In analyzing the measurements, however, it proves to be very difficult to separate the light components reflected from the various interfaces. A further disadvantage is the need to have a spectrometer, which is costly and requires a great deal of space.
It is therefore the object of the present invention to describe a method and apparatus for measuring thickness of transparent films. In paticular, the method and apparatus are intended to be suitable for transparent liquid films on a substrate or in a closed container, such as, for example, a microcuvette.
According to the present invention, a focusing aid is disposed along the path of an illumination beam of an objective in a position conjugated with the focal plane. The image formed in the focal plane is captured by an image-capturing device, such as, for example, a camera. The focusing aid is imaged sharply on the camera only when a partially reflective interface of an object is in the focal plane. The contrast of the image of the focusing aid, monitored with the camera, thus serves as a xe2x80x9cfocus indicatorxe2x80x9d. The invention makes use of this fact to determine thickness of various films.
The illumination beam is directed through the objective onto an object comprising a transparent film. The object used is, for example, a single-use microcuvette for blood analysis that contains a transparent blood film. Alternatively, it is also possible to use an object support that carries a transparent blood film.
By changing a position of the object in the Z direction relative to the objective, the position of the focal plane of the objective relative to the object changes. The values of the stop positions Zi are measured. At each stop position, a camera image is recorded and its focal score is determined, the image of the structure of the focusing aid being used as contrast indicator.
For the best image evaluation, it is advantageous if the structure on the focusing aid has a dimension of at least one bar, with the bar length being a multiple of the bar width and the bar width being a multiple of the resolution capability of the objective and the camera. Multiple bars can also be used. It is advantageous to use a cross-shaped figure having the dimensions discussed above. The cross can be adapted to different pixel widths and heights.
A blurred image yields a low focus score; a sharp image yields a high focus score. In other words, when the image of the structure of the focusing aid appears sharp in the camera image, it means that the interface of the single-use microcuvette is in the focal plane, and the focus score has a maximum.
The maxima of the focus scores are therefore determined for stop positions zi. The positions zi with maximal focus scores are assigned to the locations of the various interfaces such as, for example, the air-glass, the glass-liquid, the liquid-glass, and the glass-air interfaces in the case of a microcuvette filled with a liquid.
Various interfaces differ greatly in terms of reflectivity. Glass-air interfaces, for example, reflect light stronger than glass-liquid interfaces by a factor of about 10. Because the reflection intensities are incorporated into the calculation of the focus scores, it is possible to identify the type of an interface based on the magnitudes of the identified focus scores.
The thickness of a transparent film, e.g. of the liquid in a microcuvette, is then calculated from the difference between the positions z1, z2 corresponding to the maximal focus scores. The thickness d is calculated as d=(z1xe2x88x92z2)xc2x7nfilm, where nfilm is the refractive index of the film. Multiplying by film takes into account the optical path length modified by the refractive index of the film.
In correlating the maxima of the focus scores with the interfaces of the object the film being measured, certain preliminary information concerning the nature and/or approximate location of the interfaces and the film is utilized. This information yields further possibilities for making the correlation. For example, the following information can be used:
The displacement path selected for the object is so long that the image corresponding to the first interface is always captured, and the first maximum of the focus scores can thus be correlated with the first interface.
This is particularly advantageous for an object with a sandwich structure (i.e. having multiple material layers), for example, a microcuvette.
The interfaces of particular interest, for example, the inner walls of a microcuvette, are predefined with sufficient accuracy in terms of their expected location. These may be manufacturer""s tolerance data concerning the dimensions of the microcuvette. If, on the basis of these data, the outer interfaces are at a sufficient distance from the inner interfaces of the microcuvette, a direct correlation between the maxima of the focus scores and the inner interfaces is possible. The displacement path of the object and the number of camera images necessary for the calculation can then also be reduced. This approach offers the advantage in that the measurement times can be considerably reduced. The method is therefore particularly suitable for routine laboratory investigations.
The method of the present invention can reliably be used for measuring thickness of transparent films of all kinds. The transparent films can be:
self-supporting solid films in air or liquid;
solid or liquid films on a substrate (e.g. clinical smears on a glass slide); or
solid or liquid films on a substrate having an additional transparent covering layer (sandwich structure).
The films, the substrate, and any covering layers are preferably smooth surfaces. The interfaces of the films are preferably only partially reflective; i.e. they can have very small differences in refractive index. Even the low levels of reflection resulting from these interfaces are sufficient for automatic image evaluation using the image analysis as described by the method according to the present invention.